Tumor cells with increased immunogenicity and uses therfor

ABSTRACT

Tumor cells modified to express a T cell costimulatory molecule are disclosed. In one embodiment, the costimulatory molecule is a CD28/CTLA4 ligand, preferably a B lymphocyte antigen B7. The tumor cells of the invention can be modified by transfection with nucleic acid encoding a T cell costimulatory molecule, by using an agent which induces or increases expression of a T cell costimulatory molecule on the tumor cell surface or by coupling a T cell costimulatory molecule to the tumor cell surface. Tumor cells further modified to express MHC class I and/or class II molecules or in which expression of an MHC associated protein, the invariant chain, is inhibited are also disclosed. The modified tumor cells of the invention can be used in methods for treating-a patient with a tumor, preventing or inhibiting metastatic spread of a tumor or preventing or inhibiting recurrence of a tumor. A method for specifically inducing a CD4 +  T cell response against a tumor and a method for treating a tumor by modification of tumor cells in vivo are disclosed.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/261,101, filed Sep. 30, 2002; which is a continuation of U.S.application Ser. No. 09/996,148, filed Sep. 27, 2001; which is acontinuation application of U.S. application Ser. No. 09/450,798, filedNov. 29, 1999, now U.S. Pat. No. 6,319,708, issued Nov. 20, 2001; whichis a continuation application of U.S. application Ser. No. 09/159, 135,filed Sep. 23, 1998, now U.S. Pat. No. 6,149,905, issued Nov. 21, 2000;which is a divisional of U.S. application Ser. No. 08/147,772, filedNov. 3, 1993, now U.S. Pat. No.5,858,776, issued Jan. 12, 1999. Theentire contents each of the aforementioned applications are herebyincorporated herein by reference in their entirety.

GOVERNMENT FUNDING

Work described herein was supported under grant A121596 awarded by theNational Institutes of Health. The U.S. government therefore may havecertain rights to this invention.

BACKGROUND OF THE INVENTION

Induction of a T lymphocyte response is a critical initial step in ahost's immune response. Activation of T cells results in T cellproliferation, cytokine production by T cells and generation of Tcell-mediated effector functions. T cell activation requires anantigen-specific signal, often called a primary activation signal, whichresults from stimulation of a clonally-distributed T cell receptor(hereafter TcR) present on the surface of the T cell. Thisantigen-specific signal is usually in the form of an antigenic peptidebound either to a major histocompatibility complex (hereafter MHC) classI protein or an MHC class II protein present on the surface of anantigen presenting cell (hereafter APC). CD4+ T cells recognize peptidesassociated with class II molecules. Class II molecules are found on alimited number of cell types, primarily B cells, monocytes/macrophagesand dendritic cells, and, in most cases, present peptides derived fromproteins taken up from the extracellular environment. In contrast, CD8+T cells recognize peptides associated with class I molecules. Class Imolecules are found on almost all cell types and, in most cases, presentpeptides derived from endogenously synthesized proteins. For a reviewsee Germain, R., Nature 322, 687-691 (1986).

It has now been established that, in addition to an antigen-specificprimary activation signal, T cells also require a second, non-antigenspecific, signal to induce full T cell proliferation and/or cytokineproduction. This phenomenon has been termed costimulation. Mueller, D.L., et al., Annu. Rev. Immunol. 7, 445-480 (1989). Like theantigen-specific signal, the costimulatory signal is triggered by amolecule on the surface of the antigen presenting cell. A costimulatorymolecule, the B lymphocyte antigen B7, has been identified on activatedB cells and other APCs. Freeman, G. J., et al., J. Immunol. 139,3260-3267 (1987); Freeman, G. J., et al., J. Immunol. 143, 2714-2722(1989). Binding of B7 to a ligand on the surface of T cells providescostimulation to the T cell. Two structurally similar T cell-surfacereceptors for B7 have been identified, CD28 and CTLA-4. Aruffo, A. andSeed, B., Proc. Natl. Acad. Sci. USA 84, 8573-8577 (1987); Linsley, P.S., et al., J. Exp. Med. 173, 721-730, (1991); Brunet, J. F., et al.,Nature 328, 267-270 (1987); Brunet, J. F., et al., Immunol Rev. 103,21-36 (1988). CD28 is expressed constitutively on T cells and itsexpression is upregulated by activation of the T cell, such as byinteraction of the TcR with an antigen-MHC complex. In contrast, CTLA4is undetectable on resting T cells and its expression is induced byactivation.

A series of experiments have shown a functional role for a T cellactivation pathway stimulated through the CD28 receptor. Studies usingblocking antibodies to B7 and CD28 have demonstrated that theseantibodies can inhibit T cell activation, thereby demonstrating the needfor stimulation via this pathway for T cell activation. Furthermore,suboptimal polyclonal stimulation of T cells by phorbol ester oranti-CD3 antibodies can be potentiated by crosslinking of CD28 withanti-CD28 antibodies. Engagement of the TcR by an MHC molecule/peptidecomplex in the absence of the costimulatory B7 signal can lead to T cellanergy rather than activation. Damle, N. K., et al., Proc. Natl. Acad.Sci. USA 78, 5096-5100 (1981); Lesslauer, W., et al., Eur. J. Immunol.16, 1289-1295 (1986); Gimmi, C. D., et al., Proc. Natl. Acad. Sci. USA88, 6575-6579 (1991); Linsley, P. S., et al., J. Exp. Med. 173, 721-730(1991); Koulova, L., et al., J. Exp. Med. 173, 759-762 (1991);Razi-Wolf, Z., et al., Proc. Natl. Acad Sci. USA 89, 4210-4214 (1992).

Malignant transformation of a cell is commonly associated withphenotypic changes in the cell. Such changes can include loss or gain ofexpression of some proteins or alterations in the level of expression ofcertain proteins. It has been hypothesized that in some situations theimmune system may be capable of recognizing a tumor as foreign and, assuch, could mount an immune response against the tumor. Kripke, M., Adv.Cancer Res. 34, 69-75 (1981). This hypothesis is based in part on theexistence of phenotypic differences between a tumor cell and a normalcell, which is supported by the identification of tumor associatedantigens (hereafter TAAs). Schreiber, H., et al. Ann. Rev. Immunol. 6,465-483 (1988). TAAs are thought to distinguish a transformed cell fromits normal counterpart. Three genes encoding TAAs expressed in melanomacells, MAGE-1, MAGE-2 and MAGE-3, have recently been cloned. van derBruggen, P., et al. Science 254, 1643 -1647 (1991). That tumor cellsunder certain circumstances can be recognized as foreign is alsosupported by the existence of T cells which can recognize and respond totumor associated antigens presented by MHC molecules. Such TAA-specificT lymphocytes have been demonstrated to be present in the immunerepertoire and are capable of recognizing and stimulating an immuneresponse against tumor cells when properly stimulated in vitro.Rosenberg, S. A., et al. Science 233, 1318-1321 (1986); Rosenberg, S. A.and Lotze, M. T. Ann. Rev. Immunol. 4, 681-709 (1986).

However, in practice, tumors in vivo have generally not been found to bevery immunogenic and appear to be capable of evading immune response.This may result from an inability of tumor cells to induce Tcell-mediated immune responses. Ostrand-Rosenberg, S., et al., J.Immunol. 144, 4068-4071 (1990); Fearon, E. R., et al., Cell 60, 397-403(1990). A method for increasing the immunogenicity of a tumor cell invivo would be therapeutically beneficial.

SUMMARY OF THE INVENTION

Although most tumor cells are thought to express TAAs which distinguishtumor cells from normal cells and T cells which recognize TAA peptideshave been identified in the immune repertoire, an anti-tumor T cellresponse may not be induced by a tumor cell due to a lack ofcostimulation necessary to activate the T cells. It is known that manytumors are derived from cells which do not normally function asantigen-presenting cells, and, thus, may not trigger necessary signalsfor T cell activation. In particular, tumor cells may be incapable oftriggering a costimulatory signal in a T cell which is required foractivation of the T cell. This invention is based, at least in part, onthe discovery that tumor cells modified to express a costimulatorymolecule, and therefore capable of triggering a costimulatory signal,can induce an anti-tumor T cell-mediated immune response in vivo. This Tcell-mediated immune response is effective not only against the modifiedtumor cells but, more importantly, against the unmodified tumor cellsfrom which they were derived. Thus, the effector phase of the anti-tumorresponse induced by the modified tumor cells of the invention is notdependent upon expression of a costimulatory molecule on the tumorcells.

Accordingly, the invention pertains to methods of inducing or enhancingT lymphocyte-mediated anti-tumor immunity in a subject by use of amodified tumor cell having increased immunogenicity. In one aspect ofthe invention, a tumor cell is modified to express a T cellcostimulatory molecule on its surface. Prior to modification, the tumorcell may lack the ability to express a T cell costimulatory molecule,may be capable of expressing a T cell costimulatory molecule but fail todo so, or may express insufficient amounts of a T cell costimulatorymolecule to activate T cells. Therefore, a tumor cell can be modified byproviding a costimulatory molecule to the tumor cell surface, byinducing the expression of a costimulatory molecule on the tumor cellsurface or by increasing the level of expression of a costimulatorymolecule on the tumor cell surface. In one embodiment, the tumor cell ismodified by transfecting the cell with a nucleic acid encoding a T cellcostimulatory molecule in a form suitable for expression of the moleculeon the cell surface. Alternatively, the tumor cell is contacted with anagent which induces or increases expression of a T cell costimulatorymolecule on the cell surface. In yet another embodiment, the tumor cellis modified by chemically coupling a T cell costimulatory molecule tothe tumor cell surface.

In a preferred embodiment of the invention, tumor cells are modified toexpress a molecule which binds CD28 and/or CTLA4 on T lymphocytes. Tumorcells so modified can trigger a signal in T lymphocytes through CD28and/or CTLA4 to induce T lymphocyte proliferation and/or cytokineproduction. A preferred molecule which binds CD28 and/or CTLA4 is the Blymphocyte antigen B7. Thus, in one preferred embodiment, a tumor cellis transfected with an expression vector containing a gene encoding B7in a form suitable for expression of B7 on the cell surface.

Even when provided with the ability to trigger a costimulatory signal inT cells, modified tumor cells may still be incapable of inducinganti-tumor T cell-mediated immune responses due to a failure tosufficiently trigger an antigen-specific primary activation signal. Thiscan result from insufficient expression of MHC class I or class IImolecules on the tumor cell surface. Accordingly, this inventionencompasses modified tumor cells which provide both a T cellcostimulatory signal and an antigen-specific primary activation signal,via an antigen-MHC complex, to T cells. Prior to modification, a tumorcell may lack the ability to express one or more MHC molecules, may becapable of expressing one or more MHC molecules but fail to do so, mayexpress only certain types of MHC molecules (e.g., class I but not classII), or may express insufficient amounts of MHC molecules to activate Tcells. Thus, in one embodiment, a tumor cell is modified by providingone or more MHC molecules to the tumor cell surface, by inducing theexpression of one or more MHC molecules on the tumor cell surface or byincreasing the level of expression of one or more MHC molecules on thetumor cell surface. Tumor cells expressing a T cell costimulatorymolecule are further modified, for example, by transfection with anucleic acid encoding one or more MHC molecules in a form suitable forexpression of the MHC molecule(s) on the tumor cell surface.Alternatively, such tumor cells are modified by contact with an agentwhich induces or increases expression of one or more MHC molecules onthe cell.

In a particularly preferred embodiment, tumor cells modified to expressa T cell costimulatory molecule are further modified to express one ormore MHC class II molecules. To provide an MHC class II molecule, atleast one nucleic acid encoding an MHC class II α chain protein and anMHC class II β chain protein are introduced into the tumor cell suchthat expression of these proteins is directed to the surface of thecell. In yet another embodiment, tumor cells modified to express acostimulatory molecule are further modified to express one or more MHCclass I molecules. To provide an MHC class I molecule, at least onenucleic acid encoding an MHC class I a chain protein and a β-2microglobulin protein are introduced such that expression of theseproteins is directed to the surface of the tumor cell. Alternatively, atumor cell modified to express a costimulatory molecule can be furthermodified by contact with an agent which induces or increases theexpression of MHC molecules (class I and/or class II) on the cellsurface.

In certain situations, modified tumor cells of the invention may fail toactivate T cells because of insufficient association of TAA-derivedpeptides with MHC molecules, resulting in a lack of an antigen-specificprimary activation signal in T cells. Accordingly, the invention furtherpertains to a tumor cell modified to trigger a costimulatory signal in Tcells and in which association of TAA peptides with MHC class IImolecules is promoted in order to induce an antigen-specific signal in Tcells. This aspect of the invention is based, at least in part, on theability of an MHC class II associated protein, the invariant chain, toprevent association of endogenously derived peptides (which wouldinclude a number of TAA peptides) with MHC class II moleculesintracellularly. Thus, in one embodiment, a tumor cell modified toexpress a costimulatory molecule is further modified to promoteassociation of TAA peptides with MHC class II molecules by inhibitingthe expression of the invariant chain in the tumor cell. The tumor cellselected to be so modified can be one which naturally expresses both MHCclass II molecules and the invariant chain or can be one which expressesthe invariant chain and which has been modified to express MHC class IImolecules. Preferably, expression of the invariant chain is inhibited ina tumor cell by introducing into the tumor cell a nucleic acid which isantisense to a coding or regulatory region of the invariant chain gene.Alternatively, expression of the invariant chain in a tumor cell isprevented by an agent which inhibits expression of the invariant chaingene or which inhibits expression or activity of the invariant chainprotein.

The modified tumor cells of the invention can be used in methods forinducing an anti-tumor T lymphocyte response in a subject effectiveagainst both modified and unmodified tumor cells. For example, tumorcells can be obtained, modified as described herein to trigger acostimulatory signal in T lymphocytes, and administered to the subjectto elicit a T cell-mediated immune response. The modified tumor cells ofthe invention can also be administered to prevent or inhibit metastaticspread of a tumor or to prevent or inhibit recurrence of a tumorfollowing therapeutic treatment.

This invention also provides methods for treating a subject with a tumorby modifying tumor cells in vivo to be capable of triggering acostimulatory signal in T cells, and, if necessary, also anantigen-specific signal.

The tumor cells of the current invention modified to express both acostimulatory molecule and one or more MHC class II molecules can beused in a method for specifically inducing an anti-tumor response byCD4+ T lymphocytes in a subject with a tumor by administering themodified tumor cells to the subject. Alternatively, a CD4+ T cellresponse can be induced by modifying tumor cells in vivo to express acostimulatory molecule and one or more MHC class II molecules.

The invention also pertains to a composition of modified tumor cellssuitable for pharmaceutical administration. This composition comprisesan amount of tumor cells and a physiologically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs depicting the cell surface expression of B7 and theMHC class II molecule I-A^(k) on wild-type and transfected tumor cellsas determined by immunofluourescent staining of the cells.

DETAILED DESCRIPTION OF THE INVENTION

The induction of a T cell response requires that at least two signals bedelivered by ligands on a stimulator cell to the T cell through cellsurface receptors on the T cell. A primary activation signal isdelivered to the T cell through the antigen-specific TcR.Physiologically, this signal is triggered by an antigen-MHC moleculecomplex on the stimulator cell, although it can also be triggered byother means such as phorbol ester treatment or crosslinking of the TcRcomplex with antibodies, e.g. with anti-CD3. To induce T cellactivation, a second signal, called a costimulatory signal, is requiredby stimulation of the T cell through another cell surface molecule, suchas CD28 or CTLA4. Thus, the minimal molecules on a stimulator cellrequired for T cell activation are an MHC molecule associated with apeptide antigen, to trigger a primary activation signal in a T cell, anda costimulatory molecule to trigger a costimulatory signal in the Tcell. Engagement of the antigen-specific TcR in the absence oftriggering of a costimulatory signal can prevent activation of the Tcell and, in addition, can induce a state of unresponsiveness or anergyin the T cells.

The ability of a tumor cell to evade an immune response and fail tostimulate a T lymphocyte response against the cell may result from theinability of the cell to properly activate T cells. This inventionprovides modified tumor cells which trigger a costimulatory signal in Tcells and, thus, activate an anti-tumor T lymphocyte response.Additionally, in certain embodiments, tumor cells are modified totrigger both a primary, antigen-specific activation signal and acostimulatory signal in T cells. The modified tumor cells of theinvention display increased immunogenicity and can be used to induce orenhance a T cell-mediated immune response against a tumor. Since theeffector phase of the T cell-mediated immune response is not dependentupon expression of a costimulatory molecule by tumor cells, the Tcell-mediated immune response generated by administration of a modifiedtumor cell of the invention is effective against not only the modifiedtumor cells but also the unmodified tumor cells from which they werederived.

I. Ex Vivo Modification of a Tumor Cell to Express a CostimulatoryMolecule

The inability of a tumor cell to trigger a costimulatory signal in Tcells may be due to a lack of expression of a costimulatory molecule,failure to express a costimulatory molecule even though the tumor cellis capable of expressing such a molecule, or insufficient expression ofa costimulatory molecule on the tumor cell surface. Thus, according toone aspect of the invention, a tumor cell is modified to express acostimulatory molecule by transfection of the tumor cell with a nucleicacid encoding a costimulatory molecule in a form suitable for expressionof the costimulatory molecule on the tumor cell surface. Alternatively,the tumor cell is modified by contact with an agent which induces orincreases expression of a costimulatory molecule on the tumor cellsurface. In yet another embodiment, a costimulatory molecule is coupledto the surface of the tumor cell to produce a modified tumor cell. Theterm “costimulatory molecule” is defined herein as a molecule whichinteracts with a T cell which has received a primary activation signalto result in T cell proliferation and/or cytokine production. Preferredcostimulatory molecules include antigens on the surface of Blymphocytes, professional antigen presenting cells (e.g., monocytes,dendritic cells, Langerhans cells) and other cells which present antigento immune cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, oligodendrocytes) and which bind either CD28, CTLA4, bothCD28 and CTLA4, or other known or as yet undefined receptors on immunecells. A particularly preferred costimulatory molecule which binds CD28and/or CTLA4 is the B lymphocyte antigen B7.

The ability of a molecule to provide a costimulatory signal to T cellscan be determined, for example, by contacting T cells which havereceived a primary activation signal with the molecule to be tested anddetermining the presence of T cell proliferation and/or cytokinesecretion. T cell can be suboptimally stimulated with a primaryactivation signal, for instance by contact with immobilized anti-CD3antibodies or a phorbol ester. Following this stimulation, the T cellsare exposed to cells expressing a costimulatory molecule on theirsurface and the proliferation of the T cells and/or secretion ofcytokines, such as IL-2, by the T cells is determined. Proliferationand/or cytokine secretion will be increased by triggering of acostimulatory signal in the T cells. T cell proliferation can bemeasured, for example, by a standard ³H-thymidine uptake assay. Cytokinesecretion can be measured, for example, by a standard IL-2 assay. In thecase of a costimulatory molecule which is a CD28 ligand, the involvementof CD28 in T cell activation can be demonstrated by the use of blockingantibodies to CD28 which can inhibit T cell proliferation and/orcytokine secretion mediated by this pathway. Linsley, P. S., et al., J.Exp. Med. 173, 721-730 (1991), Gimmi, C. D., et al., Proc. Natl. Acad.Sci. USA 88:, 6575-6579 (1991), Freeman, G. J., et al., J. Exp. Med.174, 625-631, (1991).

Fragments, mutants or variants of costimulatory molecules, e.g. CD28and/or CTLA4 ligands such as B7, that retain the ability to interactwith T cells, trigger a costimulatory signal and activate T cellresponses, as evidenced by proliferation and/or cytokine production by Tcells that have received a primary activation signal, are consideredwithin the scope of the invention. A “fragment” of a costimulatorymolecule is defined as a portion of a costimulatory molecule whichretains costimulatory activity. For example, a fragment of acostimulatory molecule may have fewer amino acid residues than theentire protein. A “mutant” is defined as a costimulatory molecule havinga structural change which may enhance, diminish, not affect, but noteliminate the costimulatory activity of the molecule. For example, amutant of a costimulatory molecule may have a change in one or moreamino acid residues of the protein. A “variant” is defined as acostimulatory molecule having a modification which does not affect thecostimulatory activity of the molecule. For example, a variant of acostimulatory molecule may have altered glycosylation or may be achimeric protein of the costimulatory molecule and another protein.

A. Transfection of a Tumor Cell with a Nucleic Acid Encoding aCostimulatory Molecule

Tumor cells can be modified ex vivo to express a T cell costimulatorymolecule by transfection of isolated tumor cells with a nucleic acidencoding a costimulatory molecule in a form suitable for expression ofthe molecule on the surface of the tumor cell. The terms “transfection”or “transfected with” refers to the introduction of exogenous nucleicacid into a mammalian cell and encompass a variety of techniques usefulfor introduction of nucleic acids into mammalian cells includingelectroporation, calcium-phosphate precipitation, DEAE-dextrantreatment, lipofection, microinjection and infection with viral vectors.Suitable methods for transfecting mammalian cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)) and other laboratorytextbooks. The nucleic acid to be introduced can be, for example, DNAencompassing the gene encoding the costimulatory molecule, sense strandRNA encoding the costimulatory molecule or a recombinant expressionvector containing a cDNA encoding the costimulatory molecule. PreferredcDNAs to use are those for human and mouse B7 (Freeman, G. J., et al.,J. Exp. Med 174, 625-631 (1991); Freeman, G. J., et al., J. Immunol.143, 2714-2722 (1989)). The nucleotide sequence of the human B7 cDNA isshown in SEQ ID NO:1 and the corresponding amino acid sequence of thehuman B7 protein is shown in SEQ ID NO:2. The nucleotide sequence of themouse B7 cDNA is shown in SEQ ID NO:3 and the corresponding amino acidsequence of the mouse B7 protein is shown in SEQ ID NO:4.

The nucleic acid is “in a form suitable for expression of thecostimulatory molecule” in which the nucleic acid contains all of thecoding and regulatory sequences required for transcription andtranslation of a gene, which may include promoters, enhancers andpolyadenylation signals, and sequences necessary for transport of themolecule to the surface of the tumor cell, including N-terminal signalsequences. When the nucleic acid is a cDNA in a recombinant expressionvector, the regulatory functions responsible for transcription and/ortranslation of the cDNA are often provided by viral sequences. Examplesof commonly used viral promoters include those derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.Regulatory sequences linked to the cDNA can be selected to provideconstitutive or inducible transcription, by, for example, use of aninducible promoter, such as the metallothienin promoter or aglucocorticoid-responsive promoter. Expression of the costimulatorymolecule on the surface of the tumor cell can be accomplished, forexample, by including a native transmembrane coding sequence of themolecule, such as B7, in the nucleic acid sequence, or by includingsignals which lead to modification of the protein, such as a C-terminalinositol-phosphate linkage, that allows for association of the moleculewith the outer surface of the cell membrane.

A preferred approach for introducing nucleic acid encoding acostimulatory molecule into tumor cells is by use of a viral vectorcontaining nucleic acid, e.g. a cDNA, encoding the costimulatorymolecule. Examples of viral vectors which can be used include retroviralvectors (Eglitis, M. A., et al., Science 230, 1395-1398 (1985); Danos,O. and Mulligan, R., Proc. Natl. Acad. Sci. USA 85, 6460-6464 (1988);Markowitz, D.,. et al., J. Virol. 62, 1120-1124 (1988)), adenoviralvectors (Rosenfeld, M. A., et al., Cell 68, 143-155 (1992)) andadeno-associated viral vectors (Tratschin, J. D., et al., Mol. Cell.Biol. 5, 3251-3260 (1985)). Infection of tumor cells with a viral vectorhas the advantage that a large proportion of cells will receive nucleicacid, thereby obviating a need for selection of cells which havereceived nucleic acid, and molecules encoded within the viral vector,e.g. by a cDNA contained in the viral vector, are expressed efficientlyin cells which have taken up viral vector nucleic acid.

Alternatively, a costimulatory molecule can be expressed on a tumor cellusing a plasmid expression vector which contains nucleic acid, e.g. acDNA, encoding the costimulatory molecule. Suitable plasmid expressionvectors include CDM8 (Seed, B., Nature 329, 840 (1987)) and pMT2PC(Kaufman, et al., EMBO J. 6, 187-195 (1987)). Since only a smallfraction of cells (about 1 out of 10⁵) typically integrate transfectedplasmid DNA into their genomes, it is advantageous to transfect anucleic acid encoding a selectable marker into the tumor cell along withthe nucleic acid(s) of interest. Preferred selectable markers includethose which confer resistance to drugs such as G418, hygromycin andmethotrexate. Selectable markers may be introduced on the same plasmidas the gene(s) of interest or may be introduced on a separate plasmid.Following selection of transfected tumor cells using the appropriateselectable marker(s), expression of the costimulatory molecule on thesurface of the tumor cell can be confirmed by immunofluorescent stainingof the cells. For example, cells may be stained with a fluorescentlylabeled monoclonal antibody reactive against the costimulatory moleculeor with a fluorescently labeled soluble receptor which binds thecostimulatory molecule. Expression of the B7 costimulatory molecule canbe determined using a monoclonal antibody, 133, which recognizes B7.Freedman, A. S., et al. J. Immunol. 139, 3260-3267 (1987).Alternatively, a labeled soluble CD28 or CTLA4 protein or fusion proteinwhich binds to B7 can be used to detect expression of B7.

When transfection of tumor cells leads to modification of a largeproportion of the tumor cells and efficient expression of acostimulatory molecule on the surface of tumor cells, e.g. when using aviral expression vector, tumor cells may be used without furtherisolation or subcloning. Alternatively, a homogenous population oftransfected tumor cells can be prepared by isolating a singletransfected tumor cell by limiting dilution cloning followed byexpansion of the single tumor cell into a clonal population of cells bystandard techniques.

B. Induction or Increased Expression of a Costimulatory Molecule on aTumor Cell Surface

A tumor cell can be modified to trigger a costimulatory signal in Tcells by inducing or increasing the level of expression of acostimulatory molecule on a tumor cell which is capable of expressing acostimulatory molecule but fails to do so or which expressesinsufficient amounts of the costimulatory molecule to activate T cells.An agent which stimulates expression of a costimulatory molecule can beused in order to induce or increase expression of a costimulatorymolecule on the tumor cell surface. For example, tumor cells can becontacted with the agent in vitro in a culture medium. The agent whichstimulates expression of a costimulatory molecule may act, for instance,by increasing transcription of a costimulatory molecule gene, byincreasing translation of a costimulatory molecule mRNA or by increasingstability or transport of a costimulatory molecule to the cell surface.For example, expression of B7 can be upregulated in a cell by a secondmessenger pathway involving cAMP. Nabavi, N., et al. Nature 360, 266-268(1992). Thus, a tumor cell can be contacted with an agent, whichincreases intracellular cAMP levels or which mimics cAMP, such as a cAMPanalogue, e.g. dibutyryl cAMP, to stimulate expression of B7 on thetumor cell surface. Expression of B7 can also be induced on normalresting B cells by crosslinking cell-surface MHC class II molecules onthe B cells with an antibody against the MHC class II molecules.Kuolova, L., et al., J. Exp. Med. 173, 759-762 (1991). Similarly, atumor cell which expresses MHC class II molecules on its surface can betreated with anti-MHC class II antibodies to induce or increase B7expression on the tumor cell surface.

Another agent which can be used to induce or increase expression of acostimulatory molecule on a tumor cell surface is a nucleic acidencoding a transcription factor which upregulates transcription of thegene encoding the costimulatory molecule. This nucleic acid can betransfected into the tumor cell to cause increased transcription of thecostimulatory molecule gene, resulting in increased cell-surface levelsof the costimulatory molecule.

C. Coupling of a Costimulatory Molecule to the Surface of a Tumor Cell

In another embodiment, a tumor cell is modified to be capable oftriggering a costimulatory signal in T cells by coupling a costimulatorymolecule to the surface of the tumor cell. For example, a costimulatorymolecule, such as B7, can be obtained using standard recombinant DNAtechnology and expression systems which allows for production andisolation of the costimulatory molecule. Alternatively, a costimulatorymolecule can be isolated from cells which express the costimulatorymolecule using standard protein purification techniques. For example, B7protein can be isolated from activated B cells by immunoprecipitationwith an anti-B7 antibody such as the 133 monoclonal antibody. Theisolated costimulatory molecule is then coupled to the tumor cell. Theterms “coupled” or “coupling” refer to a chemical, enzymatic or othermeans (e.g. antibody) by which a costimulatory molecule is linked to atumor cell such that the costimulatory molecule is present on thesurface of the tumor cell and is capable of triggering a costimulatorysignal in T cells. For example, the costimulatory molecule can bechemically crosslinked to the tumor cell surface using commerciallyavailable crosslinking reagents (Pierce, Rockford Ill.). Anotherapproach to coupling a costimulatory molecule to a tumor cell is to usea bispecific antibody which binds both the costimulatory molecule and acell-surface molecule on the tumor cell. Fragments, mutants or variantsof costimulatory molecules which retain the ability to trigger acostimulatory signal in T cells when coupled to the surface of a tumorcell can also be used.

II. Additional Modification of a Tumor Cell to Express MHC Molecules

Another aspect of this invention features modified tumor cells whichexpress a costimulatory molecule and which express one or more MHCmolecules on their surface to trigger both a costimulatory signal and aprimary, antigen-specific, signal in T cells. Before modification, tumorcells may be unable to express MHC molecules, may fail to express MHCmolecules although they are capable of expressing such molecules, or mayexpress insufficient amounts of MHC molecules on the tumor cell surfaceto cause T cell activation. Tumor cells can be modified to expresseither MHC class I or MHC class II molecules, or both. One approach tomodifying tumor cells to express MHC molecules is to transfect the tumorcell with one or more nucleic acids encoding one or more MHC molecules.Alternatively, an agent which induces or increases expression of one ormore MHC molecules on tumor cells can be used to modify tumor cells.Inducing or increasing expression of MHC class II molecules on a tumorcell can be particularly beneficial for activating CD4⁺ T cells againstthe tumor since the ability of MHC class II⁺ tumor cells to directlypresent tumor peptides to CD4⁺ T cells bypasses the need forprofessional MHC class II⁺ APCs. This can improve tumor immunogenicitybecause soluble tumor antigen (in the form of tumor cell debris orsecreted protein) may not be available for uptake by professional MHCclass II⁺ APCs.

One embodiment of the invention is a modified tumor cell which expressesa costimulatory molecule and one or more MHC class II molecules on theircell surface. MHC class II molecules are cell-surface α/β heterodimerswhich structurally contain a cleft into which antigenic peptides bindand which function to present bound peptides to the antigen-specificTcR. Multiple, different MHC class II proteins are expressed onprofessional APCs and different MHC class II proteins bind differentantigenic peptides. Expression of multiple MHC class II molecules,therefore, increases the spectrum of antigenic peptides that can bepresented by an APC or by a modified tumor cell. The α and β chains ofMHC class II molecules are encoded by different genes. For instance, thehuman MHC class II protein HLA-DR is encoded by the HLA-DRα and HLA-DRβgenes. Additionally, many polymorphic alleles of MHC class II genesexist in human and other species. T cells of a particular individualrespond to stimulation by antigenic peptides in conjunction with selfMHC molecules, a phenomenon termed MHC restriction. In addition, certainT cells can also respond to stimulation by polymorphic alleles of MHCmolecules found on the cells of other individuals, a phenomenon termedallogenicity. For a review of MHC class II structure and function, seeGermain and Margulies, Ann. Rev. Immunol. 11: 403-450, 1993.

Another embodiment of the invention is a modified tumor cell whichexpresses a costimulatory molecule and one or more MHC class I moleculeson the cell surface. Similar to MHC class II genes, there are multipleMHC class I genes and many polymorphic alleles of these genes are foundin human and other species. Like MHC class II proteins, class I proteinsbind peptide fragments of antigens for presentation to T cells. Afunctional cell-surface class I molecule is composed of an MHC class I achain protein associated with a β2-microglobulin protein.

A. Transfection of a Tumor Cell with Nucleic Acid Encoding MHC Molecules

Tumor cells can be modified ex vivo to express one or more MHC class IImolecules by transfection of isolated tumor cells with one or morenucleic acids encoding one or more MHC class II α chains and one or moreMHC class II β chains in a form suitable for expression of the MHC classII molecules(s) on the surface of the tumor cell. Both an α and a βchain protein must be present in the tumor cell to form a surfaceheterodimer and neither chain will be expressed on the cell surfacealone. The nucleic acid sequences of many murine and human class IIgenes are known. For examples see Hood, L., et al. Ann. Rev. Immunol. 1,529-568 (1983) and Auffray, C. and Strominger, J. L., Advances in HumanGenetics 15, 197-247 (1987). Preferably, the introduced MHC class IImolecule is a self MHC class II molecule. Alternatively, the MHC classII molecule could be a foreign, allogeneic, MHC class II molecule. Aparticular foreign MHC class II molecule to be introduced into tumorcells can be selected by its ability to induce T cells from atumor-bearing subject to proliferate and/or secrete cytokines whenstimulated by cells expressing the foreign MHC class II molecule (i.e.by its ability to induce an allogeneic response). The tumor cells to betransfected may not express MHC class II molecules on their surfaceprior to transfection or may express amounts insufficient to stimulate aT cell response. Alternatively, tumor cells which express MHC class IImolecules prior to transfection can be further transfected withadditional, different MHC class II genes or with other polymorphicalleles of MHC class II genes to increase the spectrum of antigenicfragments that the tumor cells can present to T cells.

Fragments, mutants or variants of MHC class II molecules that retain theability to bind peptide antigens and activate T cell responses, asevidenced by proliferation and/or lymphokine production by T cells, areconsidered within the scope of the invention. A preferred variant is anMHC class II molecule in which the cytoplasmic domain of either one orboth of the α and β chains is truncated. Truncation of the cytoplasmicdomains allows peptide binding by and cell surface expression of MHCclass II molecules but prevents the induction of endogenous B7expression, which is triggered by an intracellular signal generated bythe cytoplasmic domains of the MHC class II protein chains uponcrosslinking of cell surface MHC class II molecules. Kuolova. L., etal., J. Exp. Med. 173, 759-762 (1991); Nabavi, N., et al. Nature 360,266-268 (1992). In tumor cells transfected to constitutively express B7or other costimulatory molecule, it may be desirable to inhibit theexpression of endogenous B7, for instance to restrain potentialdownregulatory feedback mechanisms. Transfection of a tumor cell with anucleic acid(s) encoding a cytoplasmic domain-truncated form of MHCclass II α and β chain proteins would inhibit endogenous B7 expression.Such variants can be produced by, for example, introducing a stop codonin the MHC class II chain gene(s) after the nucleotides encoding thetransmembrane spanning region. The cytoplasmic domain of either the αchain or the β chain protein can be truncated, or, for more completeinhibition of B7 induction, both the α and β chains can be truncated.See e.g. Griffith et al., Proc. Natl. Acad Sci. USA 85: 4847-4852,(1988), Nabavi et al., J. Immunol. 142: 1444-1447, (1989).

Tumor cells can be modified to express an MHC class I molecule bytransfection with a nucleic acid encoding an MHC class I a chainprotein. For examples of nucleic acids see Hood, L., et al. Ann. Rev.Immunol. 1, 529-568 (1983) and Auffray, C. and Strominger, J. L.,Advances in Human Genetics 15, 197-247 (1987). Optionally, if the tumorcell does not express β-2 microglobulin, it can also be transfected witha nucleic acid encoding the β-2 microglobulin protein. For examples ofnucleic acids see Gussow, D., et al., J. Immunol. 139, 3132-3138 (1987)and Parnes, J. R., et al., Proc. Natl. Acad. Sci. USA 78, 2253-2257(1981). As for MHC class II molecules, increasing the number ofdifferent MHC class I genes or polymorphic alleles of MHC class I genesexpressed in a tumor cell can increase the spectrum of antigenicfragments that the tumor cells can present to T cells.

When a tumor cell is transfected with nucleic acid which encodes morethan one molecule, for example a B7 molecule, an MHC class II α chainprotein and an MHC class II β chain protein, the transfections can beperformed simultaneously or sequentially. If the transfections areperformed simultaneously, the molecules can be introduced on the samenucleic acid, so long as the encoded sequences do not exceed a carryingcapacity for a particular vector used. Alternatively, the molecules canbe encoded by separate nucleic acids. If the transfections are conductedsequentially and tumor cells are selected using a selectable marker, oneselectable marker can be used in conjunction with the first introducednucleic acid while a different selectable marker can be used inconjunction with the next introduced nucleic acid.

The expression of MHC molecules (class I or class II) on the cellsurface of a tumor cell can be determined, for example, byimmunoflourescence of tumor cells using fluorescently labeled monoclonalantibodies directed against different MHC molecules. Monoclonalantibodies which recognize either non-polymorphic regions of aparticular MHC molecule (non-allele specific) or polymorphic regions ofa particular MHC molecule (allele-specific) can be used are known tothose skilled in the art.

B. Induction or Increased Expression of MHC Molecules on a Tumor Cell

Another approach to modifying a tumor cell ex vivo to express MHCmolecules on the surface of a tumor cell is to use an agent whichstimulates expression of MHC molecules in order to induce or increaseexpression of MHC molecules on the tumor cell surface. For example,tumor cells can be contacted with the agent in vitro in a culturemedium. An agent which stimulates expression of MHC molecules may act,for instance, by increasing transcription of MHC class I and/or class IIgenes, by increasing translation of MHC class I and/or class II mRNAs orby increasing stability or transport of MHC class I and/or class IIproteins to the cell surface. A number of agents have been shown toincrease the level of cell-surface expression of MHC class II molecules.See for example Cockfield, S. M. et al., J. Immunol. 144, 2967-2974(1990); Noelle, R. J. et al. J. Immunol. 137, 1718-1723 (1986); Mond, J.J., et al., J. Immunol. 127, 881-888 (1981); Willman, C. L., et al. J.Exp. Med., 170, 1559-1567 (1989); Celada, A. and Maki, R. J. Immunol.146, 114-120 (1991) and Glimcher, L. H. and Kara, C. J. Ann. Rev.Immunol. 10, 13-49 (1992) and references therein. These agents includecytokines, antibodies to other cell surface molecules and phorbolesters. One agent which upregulates MHC class I and class II moleculeson a wide variety of cell types is the cytokine interferon-γ. Thus, forexample, tumor cells modified to express a costimulatory molecule can befurther modified to increase expression of MHC molecules by contact withinterferon-γ.

Another agent which can be used to induce or increase expression of anMHC molecule on a tumor cell surface is a nucleic acid encoding atranscription factor which upregulates transcription of MHC class I orclass II genes. Such a nucleic acid can be transfected into the tumorcell to cause increased transcription of MHC genes, resulting inincreased cell-surface levels of MHC proteins. MHC class I and class IIgenes are regulated by different transcription factors. However, themultiple MHC class I genes are regulated coordinately, as are themultiple MHC class II genes. Therefore, transfection of a tumor cellwith a nucleic acid encoding a transcription factor which regulates MHCgene expression may increase expression of several different MHCmolecules on the tumor cell surface. Several transcription factors whichregulate the expression of MHC genes have been identified, cloned andcharacterized. For example, see Reith, W. et al., Genes Dev. 4,1528-1540, (1990); Liou, H.-C., et al., Science 247, 1581-1584 (1988);Didier, D. K., et al., Proc. Natl. Acad. Sci. USA 85, 7322-7326 (1988).

III. Inhibition of Invariant Chain Expression in Tumor Cells

Another embodiment of the invention provides a tumor cell modified toexpress a T cell costimulatory molecule and in which expression of anMHC class II-associated protein, the invariant chain, is inhibited.Invariant chain expression is inhibited to promote association ofendogenously-derived TAA peptides with MHC class II molecules to createan antigen-MHC complex. This complex can trigger an antigen-specificsignal in T cells to induce activation of T cells in conjunction with acostimulatory signal. MHC class II molecules have been shown to becapable of presenting endogenously-derived peptides. Nuchtern, J. G., etal. Nature 343, 74-76 (1990); Weiss, S. and Bogen, B. Cell 767-776(1991). However, in cells which naturally express MHC class IImolecules, the α and β chain proteins are associated with the invariantchain (hereafter Ii) during intracellular transport of the proteins fromthe endoplasmic reticulum. It is believed that Ii functions in part bypreventing the association of endogenously-derived peptides with MHCclass II molecules. Elliott, W., et al. J. Immunol. 138, 2949-2952(1987); Stockinger, B., et al. Cell 56, 683-689 (1989); Guagliardi, L.,et al. Nature (London) 343, 133-139 (1990); Bakke, O., et al. Cell 63,707-716 (1990); Lottreau, V., et al. Nature 348,600-605 (1990); Peters,J., et al. Nature 349, 669-676 (1991); Roche, P., et al. Nature 345,615-618 (1990); Teyton, L., et al. Nature 348, 39-44 (1990). Since TAAsare synthesized endogenously in tumor cells, peptides derived from themare likely to be available intracellularly. Accordingly, inhibiting theexpression of Ii in tumor cells which express Ii may increase thelikelihood that TAA peptides -will associate with MHC class IImolecules. Consistent with this mechanism, it was shown thatsupertransfection of an MHC class II⁺, Ii⁻ tumor cell with the Ii geneprevented stimulation of tumor-specific immunity by the tumor cell.Clements, V. K., et al. J. Immunol. 149, 2391-2396 (1992).

Prior to modification, the expression of Ii in a tumor cell can beassessed by detecting the presence or absence of Ii mRNA by Northernblotting or by detecting the presence or absence of Ii protein byimmunoprecipitation. A preferred approach for inhibiting expression ofIi is by introducing into the tumor cells a nucleic acid which isantisense to a coding or regulatory region of the Ii gene, which havebeen previously described. Koch, N., et al., EMBO J. 6, 1677-1683,(1987). For example, an oligonucleotide complementary to nucleotidesnear the translation initiation site of the Ii mRNA can be synthesized.One or more antisense oligonucleotides can be added to media containingtumor cells, typically at a concentration of oligonucleotides of 200μg/ml. The antisense oligonucleotide is taken up by tumor cells andhybridizes to Ii mRNA to prevent translation. In another embodiment, arecombinant expression vector is used in which a nucleic acid encodingsequences of the Ii gene in an orientation such that mRNA which isantisense to a coding or regulatory region of the Ii gene is produced.Tumor cells transfected with this recombinant expression vector thuscontain a continuous source of Ii antisense nucleic acid to preventproduction of Ii protein. Alternatively, Ii expression in a tumor cellcan be inhibited by treating the tumor cell with an agent whichinterferes with Ii expression. For example, a pharmaceutical agent whichinhibits Ii gene expression, Ii mRNA translation or Ii protein stabilityor intracellular transport can be used.

IV. Types of Tumor Cells to be Modified

The tumor cells to be modified as described herein include tumor cellswhich can be transfected or treated by one or more of the approachesencompassed by the present invention to express a costimulatorymolecule. If necessary, the tumor cell can be further modified toexpress MHC molecules or an inhibitor of Ii expression. A tumor fromwhich tumor cells are obtained can be one that has arisen spontaneously,e.g in a human subject, or may be experimentally derived or induced,e.g. in an animal subject. The tumor cells can be obtained, for example,from a solid tumor of an organ, such as a tumor of the lung, liver,breast, colon, bone etc. Malignancies of solid organs includecarcinomas, sarcomas, melanomas and neuroblastomas. The tumor cells canalso be obtained from a blood-borne (ie. dispersed) malignancy such as alymphoma, a myeloma or a leukemia.

The tumor cells to be modified include those that express MHC moleculeson their cell surface prior to transfection and those that express no orlow levels of MHC class I and/or class II molecules. A minority ofnormal cell types express MHC class II molecules. It is thereforeexpected that many tumor cells will not express MHC class II moleculesnaturally. These tumors can be modified to express a costimulatorymolecule and MHC class II molecules. Several types of tumors have beenfound to naturally express surface MHC class II molecules, such asmelanomas (van Duinen et al., Cancer Res. 48, 1019-1025, 1988), diffuselarge cell lymphomas (O'Keane et al., Cancer 66, 1147-1153, 1990),squamous cell carcinomas of the head and neck (Mattijssen et al., Int.J. Cancer 6, 95-100, 1991) and colorectal carcinomas (Moller et al.,Int. J. Cancer 6, 155-162, 1991). Tumor cells which naturally expressclass II molecules can be modified to express a costimulatory molecule,and, in addition, other class II molecules which can increase thespectrum of TAA peptides which can be presented by the tumor cell. Mostnon-malignant cell types express MHC class I molecules. However,malignant transformation is often accompanied by downregulation ofexpression of MHC class I molecules on the surface of tumor cells.Csiba, A., et al., Brit. J. Cancer 50, 699-709 (1984). Importantly, lossof expression of MHC class I antigens by tumor cells is associated witha greater aggressiveness and/or metastatic potential of the tumor cells.Schrier, P. I., et al. Nature 305, 771-775 (1983); Holden, C. A., et al.J. Am. Acad. Dermatol. 9., 867-871 (1983); Baniyash, M., et al. J.Immunol. 129, 1318-1323(1982). Types of tumors in which MHC class Iexpression has been shown to be inhibited include melanomas, colorectalcarcinomas and squamous cell carcinomas. van Duinen et al., Cancer Res.48, 1019-1025, (1988); Moller et al., Int. J. Cancer 6, 155-162, (1991);Csiba, A., et al., Brit. J. Cancer 50, 699-709 (1984); Holden, C. A., etal. J. Am. Acad. Dermatol. 9., 867-871 (1983). A tumor cell which failsto express class I molecules or which expresses only low levels of MHCclass I molecules can be modified by one or more of the techniquesdescribed herein to induce or increase expression of MHC class Imolecules on the tumor cell surface to enhance tumor cellimmunogenicity.

V. Modification of Tumor Cells In Vivo

Another aspect of the invention provides methods for increasing theimmunogenicity of a tumor cell by modification of the tumor cell in vivoto express a costimulatory molecule to trigger a costimulatory signal inT cells. In addition, tumor cells can be further modified in vivo toexpress MHC molecules to trigger a primary, antigen-specific, signal inT cells. Tumor cells can be modified in vivo by introducing a nucleicacid encoding a T cell costimulatory molecule into the tumor cells in aform suitable for expression of the costimulatory molecule on thesurface of the tumor cells. Likewise, nucleic acids encoding MHC class Ior class II molecules or an antisense sequence of the Ii gene can beintroduced into tumor cells in vivo. In one embodiment, a recombinantexpression vector is used to deliver nucleic acid encoding B7 to tumorcells in vivo as a form of gene therapy. Vectors useful for in vivo genetherapy have been previously described and include retroviral vectors,adenoviral vectors and adeno-associated viral vectors. See e.g.Rosenfeld, M. A., Cell 68, 143-155 (1992); Anderson, W. F., Science 226,401409 (1984); Friedman, T., Science 244, 1275-1281 (1989).Alternatively, nucleic acid can be delivered to tumor cells in vivo bydirect injection of naked nucleic acid into tumor cells. See e.g.Acsadi, G., et al., Nature 332, 815-818 (1991). A delivery apparatus iscommercially available (BioRad). Optionally, to be suitable forinjection, the nucleic acid can be complexed with a carrier such as aliposome. Nucleic acid encoding an MHC class I molecule complexed with aliposome has been directly injected into tumors of melanoma patients.Hoffman, M., Science 256, 305-309 (1992).

Tumor cells can also be modified in vivo by use of an agent whichinduces or increases expression of a costimulatory molecule (and, ifnecessary, MHC molecules as described herein). The agent may beadministered systemically, e.g. by intravenous injection, or,preferably, locally to the tumor cells.

VI. The Effector Phase of the Anti-Tumor T Cell-Mediated Immune Response

The modified tumor cells of the invention are useful for stimulating ananti-tumor T cell-mediated immune response by triggering anantigen-specific signal and a costimulatory signal in tumor-specific Tcells. Following this inductive, or afferent, phase of an immuneresponse, effector populations of T cells are generated. These effectorT cell populations can include both CD4+ T cells and CD8+ T cells. Theeffector populations are responsible for elimination of tumors cell, by,for example, cytolysis of the tumor cell. Once T cells are activated,expression of a costimulatory molecule is not required on a target cellfor recognition of the target cell by effector T cells or for theeffector functions of the T cells. Harding, F. A. and Allison, J. P. J.Exp. Med 177, 1791-1796 (1993). Therefore, the anti-tumor Tcell-mediated immune response induced by the modified tumor cells of theinvention is effective against both the modified tumor cells andunmodified tumor cells which do not express a costimulatory molecule.

Additionally, the density and/or type of MHC molecules on the cellsurface required for the afferent and efferent phases of a Tcell-mediated immune response can differ. Fewer MHC molecules, or onlycertain types of MHC molecules (e.g. MHC class I but not MHC class II)may be needed on a tumor cell for recognition by effector T cells thanis needed for the initial activation of T cells. Therefore, tumor cellswhich naturally express low amounts of MHC molecules but are modified toexpress increased amounts of MHC molecules can induce a T cell-mediatedimmune response which is effective against the unmodified tumor cells.Alternatively, tumor cells which naturally express MHC class I moleculesbut not MHC class II molecules which are then modified to express MHCclass II molecules car induce a T cell-mediated immune response whichincludes effector T cell populations which can eliminate the parentalMHC class I+, class II− tumor cells.

VII. Therapeutic Compositions of Tumor Cells

Another aspect of the invention is a composition of modified tumor cellsin a biologically compatible form suitable for pharmaceuticaladministration to a subject in vivo. This composition comprises anamount of modified tumor cells and a physiologically acceptable carrier.The amount of modified tumor cells is selected to be therapeuticallyeffective. The term “biologically compatible form suitable forpharmaceutical administration . . . in vivo” means that any toxiceffects of the tumor cells are outweighed by the therapeutic effects ofthe tumor cells. A “physiologically acceptable carrier” is one which isbiologically compatible with the subject. Examples of acceptablecarriers include saline and aqueous buffer solutions. In all cases, thecompositions must be sterile and must be fluid to the extent that easysyringability exists. The term “subject” is intended to include livingorganisms in which tumors can arise or be experimentally induced.Examples of subjects include humans, dogs, cats, mice, rats, andtransgenic species thereof.

Administration of the therapeutic compositions of the present inventioncan be carried out using known procedures, at dosages and for periods oftime effective to achieve the desired result. For example, atherapeutically effective dose of modified tumor cells may varyaccording to such factors as age, sex and weight of the individual, thetype of tumor cell and degree of tumor burden, and the immunologicalcompetency of the subject. Dosage regimens may be adjusted to provideoptimum therapeutic responses. For instance, a single dose of modifiedtumor cells may be administered or several doses may be administeredover time. Administration may be by injection, including intravenous,intramuscular, intraperitoneal and subcutaneous injections.

VIII. Activation of Tumor-Specific T Lymphocytes In Vitro

Another approach to inducing or enhancing an anti-tumor T cell-mediatedimmune response by triggering a costimulatory signal in T cells is toobtain T lymphocytes from a tumor-bearing subject and activate the cellsin vitro by contact with a tumor cell and a stimulatory form of acostimulatory molecule. T cells can be obtained from a subject, forexample, from peripheral blood. Peripheral blood can be furtherfractionated to remove red blood cells and enrich for or isolate Tlymophocytes or T lymphocyte subpopulations. T cells can be activated invitro by culturing the T cells with tumor cells obtained from thesubject (e.g. from a biopsy or from peripheral blood in the case ofblood-borne malignancies) together with a stimulatory form of acostimulatory molecule or, alternatively, by exposure to a modifiedtumor cell as described herein. The term “stimulatory form” means thatthe costimulatory molecule is capable of crosslinking its receptor on aT cell and triggering a costimulatory signal in T cells. The stimulatoryform of the costimulatory molecule can be, for example, a solublemultivalent molecule or an immobilized form of the costimulatorymolecule, for instance coupled to a solid support. Fragments, mutants orvariants (e.g. fusion proteins) of costimulatory molecules which retainthe ability to trigger a costimulatory signal in T cells can also beused. In a preferred embodiment, a soluble extracellular portion of B7is used to provide costimulation to the T cells. Following culturing ofthe T cells in vitro with tumor cells and a costimulatory molecule, or amodified tumor cell, to activate tumor-specific T cells, the T cells canbe administered to the subject, for example by intravenous injection.

IX. Therapeutic Uses of Modified Tumor Cells

The modified tumor cells of the present invention can be used toincrease tumor immunogenicity, and therefore can be used therapeuticallyfor inducing or enhancing T lymphocyte-mediated anti-tumor immunity in asubject with a tumor or at risk of developing a tumor. A method fortreating a subject with a tumor involves obtaining tumor cells from thesubject, modifying the tumor cells ex vivo to express a T cellcostimulatory molecule, for example by transfecting them with anappropriate nucleic acid, and administering a therapeutically effectivedose of the modified tumor cells to the subject. Appropriate nucleicacids to be introduced into a tumor cell include a nucleic acid encodinga T cell costimulatory molecule, for example a CD28 and/or CTLA4 ligandsuch as B7, alone or together with nucleic acids encoding MHC molecules(class I or class II) or Ii antisense sequences as described herein.Alternatively, after tumor cells are obtained from a subject, they canbe modified ex vivo using an agent which induces or increases expressionof a costimulatory molecule (and possibly also using agent(s) whichinduce or increase MHC molecules).

Tumor cells can be obtained from a subject by, for example, surgicalremoval of tumor cells, e.g. a biopsy of the tumor, or from a bloodsample from the subject in cases of blood-borne malignancies. In thecase of an experimentally induced tumor, the cells used to induce thetumor can be used, e.g. cells of a tumor cell line. Samples of solidtumors may be treated prior to modification to produce a single-cellsuspension of tumor cells for maximal efficiency of transfection.Possible treatments include manual dispersion of cells or enzymaticdigestion of connective tissue fibers, e.g. by collagenase.

Tumor cells can be transfected immediately after being obtained from thesubject or can be cultured in vitro prior to transfection to allow forfurther characterization of the tumor cells (e.g. determination of theexpression of cell surface molecules). The nucleic acids chosen fortransfection can be determined following characterization of theproteins expressed by the tumor cell. For instance, expression of MHCproteins on the cell surface of the tumor cells and/or expression of theIi protein in the tumor cell can be assessed. Tumors which express no,or limited amounts of or types of MHC molecules (class I or class II)can be transfected with nucleic acids encoding MHC proteins; tumorswhich express Ii protein can be transfected with Ii antisense sequences.If necessary, following transfection, tumor cells can be screened forintroduction of the nucleic acid by using a selectable marker (e.g. drugresistance) which is introduced into the tumor cells together with thenucleic acid of interest.

Prior to administration to the subject, the modified tumor cells can betreated to render them incapable of further proliferation in thesubject, thereby preventing any possible outgrowth of the modified tumorcells. Possible treatments include irradiation or mitomycin C treatment,which abrogate the proliferative capacity of the tumor cells whilemaintaining the ability of the tumor cells to trigger antigen-specificand costimulatory signals in T cells and thus to stimulate an immuneresponse.

The modified tumor cells can be administered to the subject by injectionof the tumor cells into the subject. The route of injection can be, forexample, intravenous, intramuscular, intraperitoneal or subcutaneous.Administration of the modified tumor cells at the site of the originaltumor may be beneficial for inducing local T cell-mediated immuneresponses against the original tumor. Administration of the modifiedtumor cells in a disseminated manner, e.g. by intravenous injection, mayprovide systemic anti-tumor immunity and, furthermore, may protectagainst metastatic spread of tumor cells from the original site. Themodified tumor cells can be administered to a subject prior to or inconjunction with other forms of therapy or can be administered afterother treatments such as chemotherapy or surgical intervention.

Another method for treating a subject with a tumor is to modify tumorcells in vivo to express a costimulatory molecule, alone or inconjunction with MHC molecules and/or an inhibitor of Ii expression.This method can involve modifying tumor cells in vivo by providingnucleic acid encoding the protein(s) to be expressed using vectors anddelivery methods effective for in vivo gene therapy as described in aprevious section. Alternatively, one or more agents which induce orincrease expression of a costimulatory molecule, and possibly MHCmolecules, can be administered to a subject with a tumor.

The modified tumor cells of the current invention may also be used in amethod for preventing or treating metastatic spread of a tumor orpreventing or treating recurrence of a tumor. As demonstrated in detailin one of the following examples, anti-tumor immunity induced byB7-expressing tumor cells is effective against subsequent challenge bytumor cells, regardless of whether the tumor cells of the re-exposureexpress B7 or not. Thus, administration of modified tumor cells ormodification of tumor cells in vivo as described herein can providetumor immunity against cells of the original, unmodified tumor as wellas metastases of the original tumor or possible regrowth of the originaltumor.

The current invention also provides a composition and a method forspecifically inducing an anti-tumor response in CD4⁺ T cells. CD4⁺ Tcells are activated by antigen in conjunction with MHC class IImolecules. Association of peptidic fragments of TAAs with MHC class IImolecules results in recognition of these antigenic peptides by CD4⁺ Tcells. Providing a subject with tumor cells which have been modified toexpress MHC class II molecules along with a costimulatory molecule, ormodified in vivo to express MHC class II molecules along with acostimulatory molecule, can be useful for directing tumor antigenpresentation to the MHC class II pathway and thereby result in antigenrecognition by and activation of CD4⁺ T cells specific for the tumorcells. As explained in detail in an example to follow, depletion ofeither CD4⁺ or CD8⁺ T cells in vivo, by administration of anti-CD4 oranti-CD8 antibodies, can be used to demonstrate that specific anti-tumorimmunity is mediated by a particular (e.g. CD4⁺) T cell subpopulation.

As demonstrated in Example 2, subjects initially exposed to modifiedtumor cells develop an anti-tumor specific T cell response which iseffective against subsequent exposure to unmodified tumor cells. Thusthe subject develops anti-tumor specific immunity. The generalized useof modified tumor cells of the invention from one human subject as animmunogen to induce anti-tumor immunity in another human subject isprohibited by histocompatibility differences between unrelated humans.However, use of modified tumor cells from one individual to induceanti-tumor immunity in another individual to protect against possiblefuture occurrence of a tumor may be useful in cases of familialmalignancies. In this situation, the tumor-bearing donor of tumor cellsto be modified is closely related to the (non-tumor bearing) recipientof the modified tumor cells and therefore the donor and recipient shareMHC antigens. A strong hereditary component has been identified forcertain types of malignancies, for example certain breast and coloncancers. In families with a known susceptibility to a particularmalignancy and in which one individual presently has a tumor, tumorcells from that individual could be modified to express a costimulatorymolecule and administered to susceptible, histocompatible family membersto induce an anti-tumor response in the recipient against the type oftumor to which the family is susceptible. This anti-tumor response couldprovide protective immunity to subsequent development of a tumor in theimmunized recipient.

X. Tumor-Specific T Cell Tolerance

In the case of an experimentally induced tumor, such as described inExamples 1 to 3, a subject (e.g. a mouse) can be exposed to the modifiedtumor cells of the invention before being challenged with unmodifiedtumor cells. Thus, the subject is initially exposed to TAA peptides ontumor cells together with a costimulatory molecule, which activatesTAA-specific T cells. The activated T cells are then effective againstsubsequent challenge with unmodified tumor cells. In the case of aspontaneously arising tumor, as is the case with human subjects, thesubject's immune system will be exposed to unmodified tumor cells beforeexposure to the modified tumor cells of the invention. Thus, the subjectis initially exposed to TAA peptides on tumor cells in the absence of acostimulatory signal. This situation is likely to induce TAA-specific Tcell tolerance in those T cells which are exposed to and are in contactwith the unmodified tumor cells. Secondary exposure of the subject tomodified tumor cells which can trigger a costimulatory signal may not besufficient to overcome tolerance in TAA-specific T cells which wereanergized by primary exposure to the tumor. Use of modified tumor cellsto induce anti-tumor immunity in a subject already exposed to unmodifiedtumor cells may therefore be most effective in early diagnosed patientswith small tumor burdens, for instance a small localized tumor which hasnot metastasized. In this situation, the tumor cells are confined to alimited area of the body and thus only a portion of the T cellrepertoire may be exposed to tumor antigens and become anergized.Administration of modified tumor cells in a systemic manner, forinstance after surgical removal of the localized tumor and modificationof isolated tumor cells, may expose non-anergized T cells to tumorantigens together with a costimulatory molecule, thereby inducing ananti-tumor response in the non-anergized T cells. The anti-tumorresponse may be effective against possible regrowth of the tumor oragainst micrometastases of the original tumor which may not have beendetected. To overcome widespread peripheral T cell tolerance to tumorcells in a subject, additional signals, such as a cytokine, may need tobe provided to the subject together with the modified tumor cells. Acytokine which functions as a T cell growth factor, such as IL-2, couldbe provided to the subject together with the modified tumor cells. IL-2has been shown to be capable of restoring the alloantigen-specificresponses of previously anergized T cells in an in vitro system whenexogenous IL-2 is added at the time of secondary alloantigenicstimulation. Tan, P., et al. J. Exp. Med 177, 165-173 (1993).

Another approach to generating an anti-tumor T cell response in asubject despite tolerance of the subject's T cells to the tumor is tostimulate an anti-tumor response in T cells from another subject who hasnot been exposed to the tumor (referred to as a naive donor) andtransfer the stimulated T cells from the naive donor back into thetumor-bearing subject so that the transferred T cells can mount animmune response against the tumor cells. An anti-tumor response isinduced in the T cells from the naive donor by stimulating the T cellsin vitro with the modified tumor cells of the invention. Such anadoptive transfer approach is generally prohibited in outbredpopulations because of histocompatibity differences between thetransferred T cells and the tumor-bearing recipient. However, advancesin allogeneic bone marrow transplantation can be applied to thissituation to allow for acceptance by the recipient of the adoptivelytransferred cells and prevention of graft versus host disease. First, atumor-bearing subject (referred to as the host) is prepared for andreceives an allogeneic bone marrow transplant from a naive donor by aknown procedure. Preparation of the host involves whole bodyirradiation, which destroys the host's immune system, including T cellstolerized to the tumor, as well as the tumor cells themselves. Bonemarrow transplantation is accompanied by treatment(s) to prevent graftversus host disease such as depletion of mature T cells from the bonemarrow graft, treatment of the host with immunosuppressive drugs ortreatment of the host with an agent, such as CTLA4Ig, to induce donor Tcell tolerance to host tissues. Next, to provide anti-tumor specific Tcells to the host which can respond against residual tumor cells in thehost or regrowth or metastases of the original tumor in the host, Tcells from the naive donor are stimulated in vitro with tumor cells fromthe host which have been modified, as described herein, to express acostimulatory molecule. Thus, the donor T cells are initially exposed totumor cells together with a costimulatory signal and therefore areactivated to respond to the tumor cells. These activated anti-tumorspecific T cells are then transferred to the host where they arereactive against unmodified tumor cells. Since the host has beenreconstituted with the donor's immune system, the host will not rejectthe transferred T cells and, additionally, the treatment of the host toprevent graft versus host disease will prevent reactivity of thetransferred T cells with normal host tissues.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

In Examples 1-3, mouse sarcoma cells were modified to express the T cellcostimulatory molecule B7. The following methodology was used inExamples 1 to 3.

Methods and Materials

A. Cells

SaI tumor cells were maintained as described (Ostrand-Rosenberg, S., etal., J. Immunol. 144, 4068-4071 (1990)).

B. Antibodies

The monoclonal antibody (mAb) 10-3.6, specific for I-A^(k) (Oi, V., etal. Curr. Top. Microbiol. Immunol. 81, 115-120 (1978)), was prepared andused as described. Ostrand-Rosenberg, S., et al., J. Immunol. 144:4068-4071 (1990). The B7-specific mAb 1G10 is a rat IgG2a mAb and wasused as described (Nabavi, N., et al. Nature 360, 266-268 (1992)). mAbsspecific for CD4⁺[GK1.5 (Wilde, D. B., et al. J. Immunol. 131, 2178-2183(1983))] and CD8⁺[2.43 (Sarmiento, M., et al. J. Immunol. 125, 2665-2672(1980))] were used as ascites fluid.

C. Transfections

Mouse SaI sarcoma cells were transfected as described inOstrand-Rosenberg, S., et al., J. Immunol. 144, 4068-4071 (1990). SaIcells (2×10⁶) were transfected by the calcium phosphate method (Wigleret al., Proc. Natl. Acad. Sci. USA, 76, 1373 (1979)). SaI cells weretransfected with wild-type Aα^(k) and Aβ^(k) MHC class II cDNAs(Ostrand-Rosenberg, S., et al., J. Immunol. 144, 4068-4071 (1990)),Aα^(k) and Aβ^(k) cDNAs truncated for their C-terminal 12 and 10 aminoacids, respectively (Nabavi, N., et al. J. Immunol. 142, 1444-1447(1989)), and/or B7 gene (Freeman, G. J., et al. J. Exp. Med. 174,625-631 (1991)). For transfection, the murine B7 cDNA was subcloned intothe eukaryotic expression vector dCDNAI (Invitrogen, San Diego, Calif.).Class II transfectants were cotransfected with pSV2neo plasmid andselected for resistance to G418 (400 μg/ml). B7 transfectants werecotransfected with pSV2hph plasmid and selected forhygromycin-resistance (400 μg/ml). All transfectants were cloned twiceby limiting dilution, except SaI/B7 transfectants, which were uncloned,and maintained in drug. Double transfectants were maintained in G418plus hygromycin. The numbers after each transfectant are the clonedesignation.

D. Immunofluorescence

Indirect immunofluorescence was performed as described(Ostrand-Rosenberg. S., et al., J. Immunol. 144, 4068-4071 (1990) ), andsamples were analyzed on an Epics C flow cytometer.

E. Tumor Challenges

For primary tumor challenges, autologous A/J mice were challengedintraperitoneally (i.p.) with the indicated number of tumor cells.Inoculated mice were checked three times per week for tumor growth. Meansurvival times of mice dying from their tumor ranged from 13 to 28 daysafter inoculation. Mice were considered to have died from their tumor ifthey contained a large volume of ascites fluid and tumor cells (≧5 ml)at the time of death. Mice were considered tumor-resistant if they weretumor-free for at least 60 days after tumor challenge (range, 60-120days). Tumor cells were monitored by indirect immunofluorescence forI-A^(k) and B7 expression prior to tumor-cell inoculation. For theexperiments of Table 2, autologous A/J mice were immunized i.p. with asingle inoculum of the indicated number of live tumor cells andchallenged i.p. with the indicated number of wild-type SaI cells 42 daysafter immunization. Mice were evaluated for tumor resistance orsusceptibility using the same criteria as for primary tumor challenge.

F. In Vivo T Cell Depletions

A/J mice were depleted of CD4⁺ or CD8⁺ T cells by i.p. inoculation with100 μl of ascites fluid of mAb GK1.5 (CD4⁺ specific; Wilde, D. B., etal., J. Immunol. 131, 2178-2183 (1983)) or mAb 2.43 (CD8⁺ specific;Sarmiento, M., et al., J. Immunol. 125, 2665-2672 (1980)) on days −6,−3,and −1 prior to tumor challenge, and every third day after tumorchallenge as described (Ghobrial, M., et al. Clin. Immunol.Immunopathol. 52, 486-506 (1989)) until the mice died or day 28,whichever came first. Presence or absence of tumor was assessed up today 28. Previous studies have established that A/J mice with largetumors at day 28 after injection will progress to death. This time pointwas, therefore, chosen to assess tumor susceptibility for the in vivodepletion experiments. One mouse per group was sacrificed on day 28, andits spleen was assayed by immunofluorescence to ascertain depletion ofthe relevant T cell population.

EXAMPLE 1 Coexpression of B7 Restores Tumor Immunogenicity

A mouse sarcoma cell line SaI was used in each of the examples. Themouse SaI sarcoma is an ascites-adapted class I⁺ class II⁻ tumor of A/J(H-2K^(k)A^(k) D^(d)) mice. The wild-type tumor is lethal in autologousA/J mice when administered i.p. It has previously been shown that SaIcells transfected with, and expressing, syngeneic MHC class II genes(Aα^(k) and Aβ^(k) genes; SaI/A^(k) cells) are immunologically rejectedby the autologous host, and immunization with live SaI/A^(k) cellsprotects mice against subsequent challenges with wild-type class II⁻ SaIcells (Ostrand-Rosenberg, S., et al., J. Immunol. 144, 40684071 (1990)).Adoptive transfer (Cole, G., et al. Cell. Immunol. 134, 480-490 (1991))and lymphocyte depletion studies (E. Lamousse-Smith and S.O.-R.,unpublished data) demonstrate that SaI and SaI/A^(k) rejection isdependent on CD4⁺ lymphocytes. SaI cells expressing class II moleculeswith truncated cytoplasmic domains (SaI/A^(k)tr cells), however, are aslethal as wild-type class II⁻ SaI cells, suggesting that the cytoplasmicregion of the class II heterodimer is required to induce protectiveimmunity (Ostrand-Rosenberg, S., et al. J. Immunol. 147, 2419-2422(1991)).

Up-regulation of the B7 activation molecule on APCs is triggered byintracellular signals transmitted by the cytoplasmic domain of the classII heterodimer, after presentation of antigen to CD4⁺ T helper cells(Nabavi, N., et al., Nature 360, 266-268 (1992)). Inasmuch as B7expression is normally up-regulated in vivo on SaI cells expressingfull-length class II molecules (S.B. and S.O.-R., unpublished data), itmay be that SaI/A^(k)tr cells do not stimulate protective immunitybecause they do not transmit a costimulatory signal.

To test whether B7 expression can compensate for the absence of theclass II cytoplasmic domain, SaI/A^(k)tr cells were supertransfectedwith a plasmid containing a cDNA encoding murine B7 under the control ofthe cytomegalovirus promoter and screened for I-A^(k) and B7 expressionby indirect immunofluorescrence. Wild-type SaI cells do not expresseither I-A^(k) or B7 (FIGS. 1 a and b), whereas SaI cells transfectedwith Aα^(k) and Aβ^(k) genes (SaI/A^(k) cells) express I-A^(k) (FIGS. 1d and f) and do not express B7 (FIGS. 1 c and e). SaI cells transfectedwith truncated class II genes plus the B7 gene (SaI/A^(k)tr/B7 cells)express I-A^(k) and B7 molecules (FIGS. 1 g and h). All cells expressuniform levels of MHC class I molecules (K^(k) and D^(d)) comparable tothe level of I-A^(k) in FIG. 1 h.

Antigen-presenting activity of the transfectants was tested bydetermining their immunogenicity and lethality in autologous A/J mice.As shown in Table 1, wild-type SaI cells administered i.p. at doses aslow as 10⁴ cells are lethal in 88-100% of mice inoculated within 13-28days after challenge, whereas 100 times as many SaI/A^(k) cells areuniformly rejected. Challenges with similar quantities of SaI/A^(k)trcells are also lethal; however, SaI/A^(k)tr cells that coexpress B7(SaI/A^(k)tr/B7 clones −1 and −3) are uniformly rejected. A/J micechallenged with SaI/A^(k)tr cells transfected with the B7 construct, butnot expressing detectable amounts of B7 antigen (SaI/A^(k)tr/hph cells),are as lethal as SaI/A^(k)tr cells, demonstrating that reversal of themalignant phenotype in SaI/A^(k)tr/B7 cells is due to expression of B7.SaI cells transfected with the B7 gene and not coexpressing truncatedclass II molecules (SaI/B7 cells, uncloned) are also as lethal aswild-type SaI cells, indicating the B7 expression without truncatedclass II molecules does not stimulate immunity. To ascertain thatrejection of SaI/A^(k) and SaI/A^(k)tr/B7 cells is immunologicallymediated, sublethally irradiated (900 rads; 1 rad=0.01 Gy) A/J mice werechallenged i.p. with these cells. In all cases, irradiated mice diedfrom the tumor. Thus, immunogenicity and host rejection of the MHC classII⁺ tumor cells are dependent on an intact class II molecule and thatcoexpression of B7 can bypass the requirement of the class IIintracellular domain. TABLE 1 Tumorigenicity of B7 and MHC classII-transfected SaI tumor cells Tumor Expression dose, Mice dead/miceChallenge tumor I-A^(k) B7 no. of cells tested, no./no. SaI — — 1 × 10⁶ 9/10 — — 1 × 10⁵  8/10 — — 1 × 10⁴ 7/8 SaI/A^(k) 19.6.4 A^(k) — 1 × 10⁶ 0/12 A^(k) — 5 × 10⁵ 0/5 A^(k) — 1 × 10⁵ 0/5 SaI/A^(k)tr 6.11.8 A^(k)tr— 1 × 10⁶ 12/12 A^(k)tr — 5 × 10⁵ 5/5 A^(k)tr — 1 × 10⁵  5/10SaI/A^(k)tr/B7-1 A^(k)tr B7 1 × 10⁶ 0/4 SaI/A^(k)tr/B7-3 A^(k)tr B7 1 ×10⁶ 0/5 A^(k)tr B7 4 × 10⁵ 0/5 A^(k)tr B7 1 × 10⁵ 0/5 SaI/A^(k)tr/hphA^(k)tr — 1 × 10⁶ 5/5 SaI/B7 — B7 1 × 10⁶ 5/5

EXAMPLE 2 Immunization with B7-Transfected Sarcoma Cells ProtectsAgainst Later Challenges of Wild-Type B7-Sarcoma

Activation of at least some T cells is thought to be dependent oncoexpression of B7. However, once the T cells are activated, B7expression is not required on the target T cell for recognition byeffector T cells. The ability of three SaI/A^(k)tr/B7 clones (B7-3, B7-1, and B7-2B5.F2) to immunize A/J mice against subsequent challenges ofwild-type class II⁻ B7⁻ SaI cells (Table 2) was determined. A/J micewere immunized with live SaI/A^(k)tr/B7 transfectants and 42 days laterchallenged with wild-type SaI tumor cells. Ninety-seven percent of miceimmunized with SaI/A^(k)tr/B7 transfectants were immune to ≧10⁶wild-type B7⁻ class II⁻ SaI cells, an immunity that is comparable tothat induced by immunization with Sal cells expressing full-length classII molecules. SaI/A^(k)tr/B7 cells, therefore, stimulate a potentresponse with long-term immunological memory against high-dosechallenges of malignant tumor cells. B7 expression is, therefore,critical for the stimulation of SaI-specific effector cells; however,its expression is not needed on the tumor targets once the appropriateeffector T cell populations have been generated. TABLE 2 Autologous A/Jmice immunized with SaI/A^(k)tr/B7 cells are immune to challenges ofwild-type SaI tumor SaI Mice dead/ No. of challenge dose mice testedImmunization immunizing cells no. of cells no./no. None — 1 × 10⁶ 5/5SaI/A^(k) 19.6, 4 1 × 10⁵ 1 × 10⁶ 0/5 or 10⁶ 1 × 10⁶ 6 × 10⁶ 0/5SaI/A^(k)tr/B7-3 1 × 10⁶ 6 × 10⁶ 0/5 1 × 10⁶ 1 × 10⁶ 0/5 4 × 10⁵ 1 × 10⁶0/5 1 × 10⁵ 5 × 10⁶ 0/5 SaI/A^(k)tr/B7-1 5 × 10⁵ 3 × 10⁶ 0/3 2 × 10⁵ 1 ×10⁶ 0/2 5 × 10⁴ 5 × 10⁶ 0/3 SaI/A^(k)tr/B7-2B5.E2 1 × 10⁵ 2 × 10⁶ 0/2 5× 10⁴ 2 × 10⁶ 1/7

EXAMPLE 3 Immunization with B7-Transfected Tumor Cells StimulatesTumor-Specific CD4± Lymphocytes

To ascertain that B7 is functioning through a T cell pathway in tumorrejection, we have in vivo-depleted A/J mice for CD4⁺ or CD8⁺ T cellsand challenged them i.p with SaI/A^(k) or SaIA^(k)tr/B7 cells. As shownin Table 3, in vivo depletion of CD4⁺ T cells results in hostsusceptibility to both SaI/A^(k) and SaI/A^(k)tr/B7 tumors, indicatingthat CD4⁺ T cells are critical for tumor rejection, whereas depletion ofCD8⁺ T cells does not affect SaI/A^(k)tr/B7 tumor rejection. Althoughimmunofluorescence analysis of splenocytes of CD8⁺-depleted micedemonstrates the absence of CD8⁺ T cells, it is possible that thedepleted mice contain small quantitites of CD8⁺ cells that are below ourlevel of detection. These data therefore demonstrate that CD4⁺ T cellsare required for tumor rejection but do not eliminate a possiblecorequirement for CD8⁺ T cells. TABLE 3 Tumor susceptibility of A/J micein vivo-depleted for CD4⁺ or CD8⁺ T cells Host T cell No. mice withtumor/ Tumor challenge depletion total no. mice challenged SaI/A^(k)CD4⁺ 3/5 SaI/A^(k)tr/B7-3 CD4⁺ 5/5 CD8⁺ 0/5

Previous adoptive transfer experiments (Cole, G., et al. Cell. Immunol.134, 480-490 (1991)) have demonstrated that both CD4⁺ and CD8⁺ T cellsare required for rejection of class II wild-type SaI cells. Inasmuch asrejection of SaI/A^(k) and SaI/A^(k)tr/B7 cells appears to require onlyCD4⁺ T cells, it is likely that immunization with class II⁺transfectants stimulates both CD4⁺ and CD8⁺ effector T cells; however,only the CD8⁺ effectors are required for rejection of class I⁺ II⁻ tumortargets. Costimulation by B7, therefore, enhances immunity bystimulating tumor-specific CD4⁺ helper and cytotoxic lymphocytes.

EXAMPLE 4 Determination of the Effect of Modified Tumor Cells inSubjects Previously Exposed to Unmodified Tumor Cells

In the previous examples, mice were immunized with modified tumor cellsto which they had not been previously exposed. In the case of treating asubject with a pre-existing tumor, the subject will be exposed tounmodified tumor cells for a period of time before exposure to modifiedtumor cells, and therefore the subject may become tolerized to theunmodified tumor cells.

To determine whether the modified tumor cells of the invention areeffective in overcoming tolerance and inducing an anti-tumor T cellresponse in a subject, mice are inoculated with increasing amounts ofwild-type SaI tumor cells which have been irradiated with 10,000 rads.Doses of tumor cells in the range of 1×10⁴ to 1×10⁶ cells can beinoculated. Tumor cells irradiated in this way survive for up to twomonths in the recipient mice, sufficient time for tolerance to the tumorcells to be induced in the mice. After two months exposure to thewild-type tumor cells, mice are injected simultaneously with wild-typetumor cells into the flank of one hind leg and with tumor cells modifiedto express B7 (eg. SaI/A^(K)tr/B7-1) into the flank of the opposite hindleg. As a control, mice are injected with wild-type tumor cells intoboth flanks. Tumor cell doses in the range of 1×10⁴ to 1×10⁶ cells areused for challenges. Tumor growth is assessed by measuring the size of atumor which grows at the site of injection. The ability of B7-modifiedtumor cells to induce anti-tumor immunity, and therefore overcome anypossible tolerance to the tumor cells in the mice, is determined by theability of B7-modified tumor cells injected into one flank to preventgrowth of wild-type tumor cells in the opposite flank, as compared towhen wild-type tumor cells are injected into both flanks.

Alternatively, the ability of B7-modified tumor cells to overcomepotential tolerance to unmodified tumor cells is assessed by an adoptivetransfer experiment. A mouse is injected intraperitoneally with a lowdose, e.g. 1×10⁴ cells, of wild-type SaI cells and the tumor cells areallowed to grow for three weeks, at which time the mouse is sacrificedand spleen cells from the mouse are harvested. These spleen cells areinjected intraperitoneally into a recipient, syngeneic mouse which hasbeen lethally irradiated to destroy its endogenous immune system. Theadoptively transferred spleen cells reconstitute the recipient mousewith an immune system which has been previously exposed to wild-typetumor cells. Following spleen cell transfer, the recipient mouse is thenchallenged with wild-type tumor cells injected into the flank of onehind leg and with B7-modified tumor cells injected into the flank of theopposite hind leg. Tumor cell doses in the range of 1×10⁴ to 1×10⁶ cellsare used for challenges. The ability of B7-modified tumor cells toinduce anti-tumor immunity is determined by the ability of B7-modifiedtumor cells injected into one flank to prevent the growth of wild-typetumor cells injected into the opposite flank.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for treating a subject with a tumor comprising modifyingtumor cells in vivo to express a T cell costimulatory molecule.
 2. Themethod of claim 1 wherein the T cell costimulatory molecule is a CD28and/or CTLA4 ligand.
 3. The method of claim 2 wherein the CD28 and/orCTLA4 ligand is a B lymphocyte antigen, B7.
 4. The method of claim 1wherein tumor cells are modified in vivo by delivering to the subject invivo a nucleic acid encoding a T cell costimulatory molecule in a formsuitable for expression of the T cell costimulatory molecule.
 5. Themethod of claim 4 wherein the nucleic acid is delivered to the subjectin vivo by injection of the nucleic acid in an appropriate vehicle intothe tumor.
 6. A method for treating a subject with a tumor, comprising:(a) obtaining tumor cells and T lymphocytes from the subject; (b)culturing the T lymphocytes from the subject in vitro with the tumorcells from the subject and with a stimulatory form of a T cell